Stabilized Proteins with Engineered Disulfide Bonds

ABSTRACT

The present invention relates to methods of introducing one or more cysteine residues into a polypeptide which permit the stabilization of the polypeptide by formation of at least one bond, preferably a disulfide bond, between different domains of the polypeptide. The invention also relates to polypeptides containing such introduced cysteine residue(s), nucleic acids encoding such polypeptides and pharmaceutical compositions comprising such polypeptides or nucleic acids. The invention also relates to vectors, viral particles and host cells containing such nucleic acids, and methods of using them to produce the polypeptides of the invention. Exemplified polypeptides include plasma proteins, including hepatocyte growth factor activator and plasma hyaluronin binding protein, as well as blood coagulation factors, such as Factor VIII, Factor V, Factor XII and prothrombin.

This invention was made with the assistance of funds provided by theGovernment of the United States. The government may own certain rightsin the present invention, pursuant to grants from the NationalInstitutes of Health, grant numbers R01HL21544, R37HL52246, T32HL07695and P01GM48495.

FIELD OF THE INVENTION

The present invention relates to methods of introducing one or morecysteine residues into a polypeptide which permit the stabilization ofthe polypeptide by formation of at least one bond, preferably adisulfide bond, between different domains of the polypeptide. Theinvention also relates to polypeptides containing such introducedcysteine residue(s), nucleic acids encoding such polypeptides andpharmaceutical compositions comprising such polypeptides or nucleicacids. The invention also relates to vectors, viral particles and hostcells containing such nucleic acids, and methods of using them toproduce the polypeptides of the invention.

BACKGROUND OF THE INVENTION

Many polypeptides are known which are the expression product of a singlegene. A number of these polypeptides are originally synthesized as asingle polypeptide chain, but contain multiple, independently foldeddomains, which are subject to limited proteolysis (or proteolyticcleavage(s)) in vivo that may result in separation of domains due todissociation of the cleavage products. Proteolysis resulting in theseparation of domains has been shown to alter the stability and/orenzymatic or functional activities of a variety of these proteins.Examples of these proteins include plasma proteins, such as thoseinvolved in blood coagulation.

As known in the art, blood clotting begins when platelets adhere to thewall of an injured blood vessel at a lesion site. Subsequently, in acascade of enzymatically regulated reactions, soluble fibrinogenmolecules are converted by the enzyme thrombin to insoluble strands offibrin that hold the platelets together in a thrombus. At each step inthe cascade, a protease precursor is converted to a protease thatcleaves the next protein precursor in the series. Cofactors are requiredat most of the steps. In its active form, the protein factor VIII is acofactor that is required for the activation of factor X by theprotease, activated factor IX.

Factor VIII can be activated to factor VIIIa (where “a” indicates“activated”) proteolytically by thrombin or factor Xa. In combinationwith calcium and phospholipid, factor VIIIa makes factor IXa a moreefficient activator of factor X by a mechanism which is not fullyunderstood.

People deficient in factor VIII or having antibodies against factor VIIIwho are not treated with factor VIII suffer uncontrolled internalbleeding that may cause a range of serious symptoms, from inflammatoryreactions in joints to early death. Severe hemophiliacs, who numberabout 10,000 in the United States, can be treated with infusion offactor VIII, which will restore the blood's normal clotting ability ifadministered with sufficient frequency and concentration.

Several preparations of human plasma-derived or recombinant factor VIIIof varying degrees of purity are available commercially for thetreatment of hemophilia A. These include a partially-purified factorVIII derived from the pooled blood of many donors that is heat- anddetergent-treated for viruses but contains a significant level ofantigenic proteins; a monoclonal antibody-purified factor VIII that haslower levels of antigenic impurities and viral contamination; andrecombinant human factor VIII.

Hemophiliacs require daily replacement of factor VIII to prevent thedeforming hemophilic arthropathy that occurs after many years ofrecurrent hemorrhages into the joints. However, supplies of factor VIIIconcentrates have never been plentiful enough for treating hemophiliacsadequately because of problems in commercial production and therapeuticuse. For example, the commonly used plasma-derived factor VIII isdifficult to isolate and purify, is immunogenic, and requires treatmentto remove the risk of infectivity from AIDS and hepatitis viruses.Porcine factor VIII may also present an alternative, however alimitation of porcine factor VIII is the development of inhibitoryantibodies to it after one or more infusions.

Activated factor VIII (FVIIIa) is thermodynamically unstable underphysiological conditions due to the tendency of the A2 domain todissociate from the rest of the complex. In other words, activated FVIIIspontaneously becomes inactive. If this dissociation could be preventedin pharmacological preparations of FVIII or FVIIIa, administration thatis less frequent and/or of lower concentration, could be realized. Thiscould result in a number of benefits such as cost savings, decreased useof medical personnel, and improved lifestyle for hemophiliacs.

Another plasma protein besides factor VIII is prothrombin. As part ofthe coagulation cascade, prothrombin is converted to thrombin by theaction of the prothrombinase complex (FXa, FVa, and Ca²⁺). In humanprothrombin, this conversion involves cleavages at Arg271 and Arg284,between the F2 domain and the thrombin A chain, and at Arg320, betweenthe A and B chains (human numbering system). In vivo, prothrombinasefirst cleaves prothrombin at Arg320, producing meizothrombin. Freemeizothrombin is an unstable intermediate, and autolysis at theArg155-Ser156 bond rapidly removes the F1 domain to generatemeizothrombin (des F1), which slowly converts to thrombin via thecleavages at Arg271 and Arg284. In the presence of thrombomodulin andphosphatidylserine/phosphatidylcholine phospholipid vesicles (PCPS),meizothrombin and meizothrombin (des F1) are better activators ofprotein C than thrombin (41, 42).

An additional plasma protein is factor V. Human coagulation factor V(FV) is a 330,000 MW protein, which is composed of six domains of threetypes in the order A1-A2-B-A3-C1-C2 (4). FV is cleaved by thrombin toremove most of the B domain and produce activated FV (FVa). Human FVa iscomposed of a heavy chain (A1-A2, residues 1-709) and a light chain(A3-C1-C2, residues 1546-2196), which form a non-covalent complex (5).FVa is the nonenzymatic cofactor for factor Xa (FXa) in theprothrombinase complex, which converts prothrombin to thrombin, in thepresence of negatively charged phospholipids (6). Inactivation of FVa isa complex process involving APC (activated Protein C) cleavages of FVaat Arg506, Arg306 and Arg679. Cleavage at Arg506 is faster than cleavageat Arg306, and it only partially inactivates FVa while cleavage atArg306 completely inactivates FVa and causes dissociation of the A2domain fragments (7-10). Fully inactive FVa loses the ability to bind toFXa (11).

Still another plasma protein is factor XII. Human FXII is a single-chainprotein with a MW of 76,000 and 596 amino acids. It contains, in orderfrom N-terminus to C-terminus fibronectin type II domain, EGF domain,fibronectin type I domain, EGF domain, Kringle domain, trypsin-likeserine protease domain. At least two forms of activated factor XII(FXIIa) exist. αFXIIa is formed by cleavage of the bond followingArg353, generating a two chain molecule comprised of a heavy chain (353residues) and a light chain (243 residues) held together by a disulfidebond. Further cleavage results in FXIIa (FXIIa fragment). This is theresult of cleavage at Arg334 and Arg343, resulting in two polypeptidechains (9 and 243 residues) held together by a disulfide bond (43, 44).The bulk of the N-terminal heavy chain fragment is no longer associated.Negative surface/membrane binding is mediated through this heavy chainso the resulting FXIIa fragment no longer binds to surfaces but it isstill catalytically active.

The protein HGFA (hepatocyte growth factor activator) has the samedomain structure as FXII (45) and is also activated by proteolyticcleavage, in this case, only one cleavage by thrombin at Arg407 (46),homologous to Arg353 in FXII. But further cleavage by kallikrein atArg372 also results in release of the N-terminal heavy chain, which, asin FXII, is involved in surface binding (47). As known in the art, HGFAactivates hepatocyte growth factor (HGF) within injured tissues whereHGF plays roles in tissue repair via a mitogenic activity towards avariety of cell types.

Another FXII-like polypeptide is known by two names: PHBP (plasmahyaluronin binding protein) (48) and FVII activating protease (49). PHBPis a serine protease and is homologous to HGFA though the domainstructure is not exactly the same (49, 50). This protein activates FVII,uPA, and tPA in experimental systems, but the physiological role has notbeen established (49, 50).

SUMMARY OF THE INVENTION

According to embodiments of the present invention, one may engineer intoa polypeptide one or more cysteine residues to permit formation of abond, such as a disulfide bond, between two or more of the polypeptide'sdomains. Placement of such disulfide bond(s) allows one to achieveresults such as polypeptide stabilization. Such stabilization can resultin the prolonged retention of desired activities of the undissociatedpolypeptide or the avoidance of undesired activities of thedisassociated polypeptide.

Preferred polypeptides useful in the invention are those which aresynthesized in nature as a single polypeptide chain, generally as theexpression product of a single gene, and which contain multiple,independently folded domains which are subject to limited proteolysisthat may result in separation of domains due to dissociation. Examplesof such polypeptides include plasma proteins, including hepatocytegrowth factor activator and plasma hyaluronin binding protein, as wellas blood coagulation factors, such as Factor VIII, Factor V, Factor XIIand prothrombin.

Mutant polypeptides of the invention (i.e., those polypeptides intowhich one or more cysteine(s) have been introduced) include not onlythose in which the domains which are linked are synthesized from asingle nucleic acid sequence (e.g., from a single gene, cDNA, orsynthetic or semi-synthetic coding sequence), but also those in whichthe domains which are linked are synthesized from distinct (or separate)nucleic acid sequences (e.g., from sequences encoding polypeptidescomprising each of the linked domains, which sequences may or may not bepresent on a contiguous nucleic acid molecule). In the latter case, thedomains may be joined together after synthesis, either in vivo or invitro.

Preferred mutant polypeptides of the invention are those which haveincreased stability and/or retain desirable enzymatic or functionalactivities for a longer period of time as compared to the correspondingunmutated polypeptide.

One aspect of the invention relates to a method of stabilizing apolypeptide which is the product of a single gene in nature byintroducing one or more cysteines comprising the steps of: (a) obtainingor creating a three-dimensional structure of the polypeptide; (b)predicting one or more sites for the introduction of one or morecysteines based on the three dimensional structure; and (c) creating oneor more mutants of said polypeptide by introducing one or more cysteinesat one or more of the predicted sites; wherein the introduction of saidone or more cysteines permits the formation of at least oneintramolecular, interdomain disulfide bridge which increases thestability of the mutant polypeptide as compared to that of thepolypeptide which does not contain said introduced one or morecysteines.

Another aspect of the invention relates to a polypeptide which is theproduct of a single gene in nature which has been mutated by introducingat least one cysteine, wherein the introduction of said cysteine permitsthe formation of at least one intramolecular, interdomain disulfidebridge with another cysteine, which increases the stability of themutant polypeptide as compared to that of the polypeptide which does notcontain said introduced cysteine.

Another aspect of the invention relates to compositions comprising thepolypeptides of the invention, including pharmaceutical compositionscomprising the polypeptides of the invention and a pharmaceuticallyacceptable carrier.

The invention also relates to nucleic acids coding for the polypeptidesof the invention, including vectors containing such nucleic acids. Theinvention also relates to viral particles containing such nucleic acidsand/or vectors. The invention also relates to host cells containing suchnucleic acids, vectors, and viral particles. The invention also relatesto compositions (including pharmaceutical compositions) which containthe nucleic acids, vectors, viral particles and/or host cells of theinvention.

The invention also relates to methods of treating individuals with thepolypeptides, nucleic acids, vectors, viral particles or host cells ofthe invention and/or pharmaceutical compositions thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of recombinant B domain-deleted FV molecules. FIG.1A is a schematic of the primary sequence of FVΔB (B-domain deletedhuman Factor V) with the locations of the different domains indicated.FIG. 1B is a schematic showing activated FVΔB (FVa), a heterodimer ofthe N-terminal heavy chain and the C-terminal light chain associated inthe presence of Ca²⁺ ions. Arrows indicate sites of cleavage in FVa byAPC. FIG. 1C is a schematic showing the cleavage fragments produced uponinactivation of FVa (FVai) by APC, and further shows the sites ofcysteine mutations that did (His609-Glu1691) and did not (Leu238-Gln590)result in disulfide bond formation.

FIG. 2. Immunoblots of various FVa and FVai mutants. (A) Immunoblotdeveloped with an anti-FV light chain monoclonal antibody. Samples inlanes 1 through 6 were not reduced and those in lanes 7 through 12 werereduced. Lanes 1 and 7, 2183A-FVa; lanes 2 and 8, 2183A-FVai; lanes 3and 9, A2-SS-A3-FVa; lanes 4 and 10, A2-SS-A3-FVai; lanes 5 and 11,Q506-A2-SS-A3-FVa,; lanes 6 and 12, Q506-A2-SS-A3-FVai. (B) Immunoblotsdeveloped with anti-FV heavy chain polyclonal antibodies. Lane 1,non-reduced A2-SS-A3-FVa; lane 2, non-reduced A2-SS-A3-FVai; lane 3,reduced A2-SS-A3-FVa; lane 4, reduced A2-SS-A3-FVai; lane 5, non-reducedQ506-A2-SS-A3-FVa; lane 6, non-reduced Q506-A2-SS-A3-FVai; lane 7,reduced Q506-A2-SS-A3-FVa; lane 8, reduced Q506-A2-SS-A3-FVai. Bandpositions for cross-linked and non cross-linked fragments are indicatedon the right side of each blot. LC=light chain, HC=heavy chain, A1=A1domain, A2=A2 domain, A2c=C-terminal fragment of the A2 domain (residues507-679). Molecular weight marker positions (kDa, Novex SeeBluestandards) are indicated on the left side.

FIG. 3 is a schematic illustrating the prevention of dissociation of theA2 domain from heterotrimeric Factor VIIIa by introduction of adisulfide bond between the A2 and A3 domains, or the A2 and A1 domains,of FVIIIa.

FIG. 4 is a schematic showing the expected action of APC upon mutantFVIIIa containing both a disulfide bridge between cysteine residuesintroduced at positions corresponding to Met 662-Asp 1828 in one mutantor Tyr 664-Thr 1826 in another mutant, and showing the APC cleavagesites at residues Arg 336 and Arg 562.

FIG. 5. Stability of Double Cysteine mutants of Factor VIIIa.Recombinant wildtype and double cysteine mutants of FVIIIa were assayedover time for activity in an APTT assay. FVIIIa species as indicated:wildtype (+), C662-C1828 (Δ), C664-C1826 (∘). At the start time about500 mU/mL FVIII was treated with 5.4 nM thrombin and then after oneminute hirudin was added to 1 U/mL to inactivate the thrombin. Thensamples were removed at indicated times and assayed for remaining FVIIIaactivity in the APTT assay.

FIG. 6 is a schematic illustrating the introduction of a disulfide bondinto human prothrombin to stabilize its meizothrombin or meizothrombin(des F1) form, preventing the conversion of meizothrombin ormeizothrombin (des F1) to alpha-thrombin (α-IIa). Legend: GLA, Gladomain; Kr.1, kringle 1 domain; Kr.2, kringle 2 domain; Meizo-IIa,meizothrombin. Prothrombin and Meizo-IIa are shown with an introduceddisulfide bond between the Kringle 2 domain and the protease domainformed from the introduction of a cysteine at a residue N-terminal toits cleavage site at residue 271 in the Kringle 2 domain and theintroduction of a cysteine at a residue C-terminal to the cleavage siteat residue 320 in the protease domain. The disulfide bond betweencysteine residues 293 and 439 is present in the naturally occurringprotein.

FIG. 7 is a description of the accession numbers and related referencesused as a source for the amino acid sequences, with notes concerning thenumbering system for Factor VIII, Factor V, Prothrombin, Factor XII,HGFA and PHBP mutants described in the examples herein.

FIG. 8. Webpages from SwissProt Accession #P00451 containing amino acidsequence of human Factor VIII and related information.

FIG. 9. Webpages from SwissProt Accession #P12259 containing amino acidsequence of human Factor V and related information.

FIG. 10. Webpages from SwissProt Accession #P00734 containing amino acidsequence of human Prothrombin and related information.

FIG. 11. Webpages from SwissProt Accession #P00748 containing amino acidsequence of human Factor XII and related information.

FIG. 12. Webpages from SwissProt Accession #Q04756 containing amino acidsequence of human HGFA and related information.

FIG. 13. Webpages from PIR Accession #JC4795 containing amino acidsequence of human PHBP and related information.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only,and are not restrictive of the invention, as claimed. The accompanyingdrawings, which are incorporated in and constitute a part of thespecification, illustrate an embodiment of the invention and, togetherwith the description, serve to explain the principles of the invention.

General Procedure

According to embodiments of the present invention, one may engineer intoa polypeptide, such as the polypeptide product of a single gene, one ormore cysteine residues which permit the formation of a disulfide bondbetween two or more of the polypeptide's domains. Placement of suchcysteine(s), with their resultant, disulfide bonds allows one to achieveresults such as polypeptide stabilization. It is noted that, in someembodiments, the present invention may also be used to place a disulfidebond within a single domain of a polypeptide, between two differentpolypeptides, and the like.

As a first step, a structure of the polypeptide of interest is obtainedor created. This may be an x-ray crystal structure, an NMR-derivedstructure, a three-dimensional structure based on homology modeling,neutron diffraction or the like.

Next, an algorithm which predicts sites for the introduction ofdisulfide bridges by placement of cysteines may be applied to astructure of the polypeptide of interest. This may be done, for example,by using the computer program MODIP which employs the algorithm ofSowdhamini (19). MODIP predicts sites for the introduction of disulfidebridges, and provides grades (A, B, C) for each prediction. Grade Asites are those predicted to be most optimal for the establishment ofdisulfide bridges, while grade B and grade C sites are progressivelyless ideal. Said differently, grade A disulfide bridges satisfy definedstereochemical criteria while grade C disulfides satisfy fewer of thestereochemical criteria. It is specifically noted that other algorithmsand/or computer programs, such as the algorithm of Pabo (18) or Hazes(56) may be used. In other embodiments, predictions for the introductioncysteines in order to establish disulfide bridges may be made by othermethods such as by visual inspection.

Of the sites predicted, one may choose a number of the most ideal sitesfor further investigation.

Visual inspection of the chosen sites may be performed usingcomputational graphics analysis. Based on this visual inspection,certain sites may be eliminated from further consideration. For each ofthe chosen sites remaining in consideration after visual inspection, amodified structural model including a disulfide bond at the chosen sitemay be created. This may be done using computer programs, such as theXfit computer program, for example, with refinement being provided byanother computer program, for example, the X-PLOR computer program usingthe Charm22 all atoms force field.

After refinement, the modeled disulfide bonds may be analyzed foroptimal disulfide geometry. Those sites with the best geometry forformation of disulfide bonds, and perhaps the lowest Van Der Waals gasphase energies, may be chosen for attempts to introduce one or morecysteine residues which permit the formation of one or more disulfidebonds. Cysteine residues may be introduced into a polypeptide usingtechniques well known in the art such as, for example, recombinanttechniques such as site directed mutagenesis of a nucleic acid encodingthe polypeptide of interest. Nucleic acids encoding polypeptides of theinvention may also be made by synthetic or semi-synthetic methods. Forexample, the nucleic acid encoding the polypeptide of the invention canbe synthesized directly using overlapping synthetic deoxynucleotides(see, e.g., Edge et al., Nature 292:756 (1981); Nambairet al., Science223:1299 (1984); Jay et al., J. Biol. Chem. 259:6311 (1984); or by usinga combination of polymerase chain reaction generated DNAs or cDNAs andsynthesized oligonucleotides. The nucleic acids of the invention can bepresent in, or inserted into an expression vector containing anappropriate promoter region operably linked to the sequence encoding thepolypeptide of the invention and an appropriate terminator signal.Afterwards, vector purification, and transfection procedures known inthe art may be performed. Next, stable clones may be selected andcollected using methods known in the art. Produced polypeptides may thenbe quantified by activity and by immunoblots so as to confirm the properplacement of the disulfide bond(s) in the polypeptide of interest andthe yields thereof.

Nucleic acids encoding the polypeptides of the invention can beexpressed in the native host cell or organism or in a different cell ororganism. The nucleic acids can be introduced into a vector such as aplasmid, cosmid, phage, virus or mini-chromosome and inserted into ahost cell or organism by methods well known in the art. In general, thenucleic acids or vectors containing these nucleic acids can be utilizedin any cell, either eukaryotic or prokaryotic, including mammalian cells(e.g., human (e.g., K293, HeLa), monkey (e.g., COS), rabbit (e.g.,rabbit reticulocytes), rat, hamster (e.g., CHO and baby hamster kidneycells) or mouse cells (e.g., L cells), plant cells, yeast cells, insectcells or bacterial cells (e.g., E. coli). The vectors which can beutilized to clone and/or express these nucleic acids encoding thepolypeptide are the vectors which are capable of replicating and/orexpressing the nucleic acids in the host cell in which the nucleic acidsare desired to be replicated and/or expressed. See, e.g., F. Ausubel etal., Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley-Interscience (1992) and Sambrook et al. (1989) forexamples of appropriate vectors for various types of host cells. Thenative promoters for such genes can be replaced with strong promoterscompatible with the host into which the nucleic acid encoding thepolypeptide of the invention is inserted. These promoters may beinducible. The host cells containing these nucleic acids can be used toexpress large amounts of the polypeptides of the invention useful inenzyme preparations, pharmaceuticals, diagnostic reagents, andtherapeutics. The polypeptides of the invention may also be made intransgenic plants or animals using methods known in the art.

If the genes which naturally encode the polypeptides of the inventioncontain inhibitory/instability regions (see, e.g., WO 93/20212)less-preferred codons may be altered to more-preferred codons. Ifdesired, however, (e.g., to make an AT-rich region more GC-rich),more-preferred codons can be altered to less-preferred codons.Optionally, only the most rarely used codons (identified from publishedcodon usage tables, such as in T. Maruyama et al., Nucl. Acids Res.14(Supp):r151-197 (1986)) may be replaced with preferred codons, oralternatively, most or all of the rare codons can be replaced withpreferred codons. Generally, the choice of preferred codons to use willdepend on the codon usage of the host cell in which the altered gene isto be expressed. Note, however, that the substitution of more-preferredcodons with less-preferred codons is also functional.

As noted above, coding sequences are chosen on the basis of the geneticcode and, preferably on the preferred codon usage in the host cell ororganism in which the nucleic acid encoding a polypeptide of thisinvention is to be expressed. In a number of cases the preferred codonusage of a particular host or expression system can be ascertained fromavailable references (see, e.g., T. Maruyama et al., Nucl. Acids Res.14(Supp):r151-197 (1986), in which the number of times the codon appearsin genes per 1000 codons is listed in parentheses next to the codon), orcan be ascertained by other methods (see, e.g., U.S. Pat. No. 5,082,767entitled “Codon Pair Utilization”, issued to G. W. Hatfield et al. onJan. 21, 1992). Preferably, sequences will be chosen to optimizetranscription and translation as well as mRNA stability so as toultimately increase the amount of polypeptide produced. Selection ofcodons is thus, for example, guided by the preferred use of codons bythe host cell and/or the need to provide for desired restrictionendonuclease sites and could also be guided by a desire to avoidpotential secondary structure constraints in the encoded mRNAtranscript. Potential secondary structure constraints can be identifiedby the use of computer programs such as the one described in M. Zuckeret al., Nucl. Acids Res. 9:133 (1981). More than one coding sequence maybe chosen in situations where the codon preference is unknown orambiguous for optimum codon usage in the chosen host cell or organism.However, any correct set of codons would encode the desired protein,even if translated with less than optimum efficiency. Example III ofSeed et al., U.S. Pat. No. 6,114,148, describes a synthetic Factor VIIIgene (encoding B-domain deleted Factor VIII), with altered codon usagewhich increases the expression of the encoded Factor VIII polypeptide.

It is also anticipated that inhibitory/instability sequences can bemutated such that the encoded amino acids are changed to contain one ormore conservative or non-conservative amino acids yet still provide fora functionally equivalent protein. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity which acts as a functional equivalent, resulting in aneutral substitution in the amino acid sequence. Substitutes for anamino acid within the sequence may be selected from other members of theclass to which the amino acid belongs. For example, the nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

Nucleic acids for genes altered by the methods of the invention orconstructs containing said nucleic acids may also be used for in-vivo orin-vitro gene replacement. For example, nucleic acid which produces apolypeptide without the introduced cysteine residue(s) can be replacedin situ with a nucleic acid that has been modified by the method of theinvention in situ to ultimately produce a polypeptide with increasedstability as compared to the originally encoded polypeptide. Such genereplacement might be useful, for example, in the development of agenetic therapy.

Vectors include retroviral vectors and also include direct injection ofDNA into muscle cells or other receptive cells, resulting in theefficient expression of the polypeptide of the invention, using thetechnology described, for example, in Wolff et al., Science247:1465-1468 (1990), Wolff et al., Human Molecular Genetics1(6):363-369 (1992) and Ulmer et al., Science 259:1745-1749 (1993). Seealso, for example, WO 96/36366 and WO 98/34640.

The polypeptides, nucleic acids, vectors, vector particles and/or hostcells of the invention can be isolated and purified by methods known inthe art and can be used in pharmaceutical compositions and/or therapiesas described further below.

Pharmaceutical Compositions

The pharmaceutical compositions of this invention contain apharmaceutically and/or therapeutically effective amount of at least onepolypeptide, or nucleic acid encoding a polypeptide, of this invention.In one embodiment of the invention, the effective amount of polypeptideper unit dose is an amount sufficient to prevent, treat or protectagainst the effects of a deficiency, or anticipated deficiency, in thecorresponding natural polypeptide. The effective amount of polypeptideper unit dose depends, among other things, on the species of mammaltreated, the body weight of the mammal and the chosen inoculationregimen, as is well known in the art.

Preferably, the route of inoculation of the peptide will be subcutaneousor intravenous. The dose is administered at least once.

The term “unit dose” refers to physically discrete units suitable asunitary dosages for mammals, each unit containing a predeterminedquantity of active material (e.g., polypeptide, or nucleic acid)calculated to produce the desired effect in association with anyaccompanying diluent.

The polypeptides or nucleic acids of the invention are generallyadministered with a physiologically acceptable carrier or vehicletherefor. A physiologically acceptable carrier is one that does notcause an adverse physical reaction upon administration and one in whichthe polypeptides or nucleic acids are sufficiently soluble and retaintheir activity to deliver a therapeutically effective amount of thecompound. The therapeutically effective amount and method ofadministration of a polypeptide or nucleic acid of the invention mayvary based on the individual patient, the indication being treated andother criteria evident to one of ordinary skill in the art. Atherapeutically effective amount of a polypeptide or nucleic acid of theinvention is one sufficient to attenuate the dysfunction without causingsignificant adverse side effects. The route(s) of administration usefulin a particular application are apparent to one of ordinary skill in theart.

Routes of administration of the polypeptides and nucleic acids of theinvention include, but are not limited to, parenteral, and directinjection into an affected site. Parenteral routes of administrationinclude but are not limited to intravenous, intramuscular,intraperitoneal and subcutaneous. The route of administration of thepolypeptides of the invention is typically parenteral.

The present invention includes compositions of the polypeptides andnucleic acids described above, suitable for parenteral administrationincluding, but not limited to, pharmaceutically acceptable sterileisotonic solutions. Such solutions include, but are not limited to,saline and phosphate buffered saline for nasal, intravenous,intramuscular, intraperitoneal, subcutaneous or direct injection into ajoint or other area.

A system for sustained delivery of the polypeptide or nucleic acid ofthe invention may also be used. For example, a delivery system based oncontaining a polypeptide in a polymer matrix of biodegradablemicrospheres may be used (57). One such polymer matrix includes thepolymer poly(lactide-co-glycolide) (PLG). PLG is biocompatible and canbe given intravenously or orally. Following injection of themicrospheres into the body, the encapsulated polypeptide is released bya complex process involving hydration of the particles and drugdissolution. The duration of the release is mainly governed by the typeof PLG polymer used and the release of modifying excipients (44).

The polypeptides and nucleic acids of the present invention are intendedto be provided to the recipient subject in an amount sufficient toprevent, or attenuate the severity, extent or duration of thedeleterious effects of a deficiency, or anticipated deficiency, in thecorresponding natural polypeptide.

The administration of the agents including polypeptide and nucleic acidcompositions of the invention may be for either “prophylactic” or“therapeutic” purpose. When provided prophylactically, the agents areprovided in advance of any symptom. The prophylactic administration ofthe agent serves to prevent or ameliorate any subsequent deleteriouseffects of the deficiency, or anticipated deficiency in thecorresponding natural polypeptide. When provided therapeutically, theagent is provided at (or shortly after) the onset of a symptom of thedeficiency or anticipated deficiency. The agent of the present inventionmay, thus, be provided either prior to the anticipated deficiency (so asto attenuate the anticipated severity, duration or extent of diseasesymptoms) or after the deficiency, and its resultant symptoms havemanifested themselves.

Also envisioned are therapies based upon vectors and viral particles,such as viral vectors and viral particles containing nucleic acidsequences coding for the polypeptides described herein. These molecules,developed so that they do not provoke a pathological effect, willproduce the encoded polypeptides of the invention.

Factor VIII Preparations

The isolation and purification of porcine and human plasma-derivedfactor VIII and human recombinant factor VIII have been described in theliterature. See, e.g., Fulcher, C. A., and T. S. Zimmerman, 79 Proc.Nat'l. Acad. Sci. U.S.A. 1648-1652 (1982); Toole, J. J., et al., 312Nature 342-347 (1984) (Genetics Institute); Gitschier, J., et al., 312Nature 326-330 (1984) (Genentech); Wood, W. I., et al., 312 Nature330-337 (1984) (Genentech); Vehar, G. A., et al., 312 Nature 337-342(1984) (Genentech); Fass, D. N., et al., 59 Blood 594 (1982); Toole, J.J., et al., 83 Proc. Nat'l. Acad. Sci. U.S.A. 5939-5942 (1986);Boedeker, B. G., Semin. Thromb. Hemost. 27(4):385-94 (Aug. 2001). Twopreparations of full-length recombinant factor VIII which were licensedfor use in humans in the early 1990s are described, e.g., in Schwartz RS, et al., N Engl J Med 323:1800-5 (1990); Lusher J M, et al., N Engl JMed 328:453-9 (1993); Bray G L, et al., Blood 83:2428-35 (1994); andWhite G C II, et al., Thromb. Haemost 77:660-7 (1997).

B-domain deleted Factor VIII, which lacks the B domain of thefull-length protein but retains coagulant activity, and which has beenlicensed for use in humans is described, e.g., in Osterbert T, et al.,Pharm Res 14:892-8 (1997); Lusher J M, et al., Blood 96:266a (2000)(abstract); and Almstedt et al., U.S. Pat. No. 5,661,008.

Hybrid human/porcine factor VIII has also been described in theliterature. See, e.g., U.S. Pat. No. 6,180,371.

The classic definition of factor VIII is that substance present innormal blood plasma that corrects the clotting defect in plasma derivedfrom individuals with hemophilia A. As used herein, factor VIII refersto a molecule which has the procoagulant properties of plasma-derivedfactor VIII or activated factor VIII. Thus, the term factor VIII, asused herein, includes a modified or truncated form of natural orrecombinant factor VIII which retains the procoagulant properties offactor VIII or activated factor VIII. Thus, factor VIII, as used herein,includes the uncleaved precursor factor VIII molecule, as well as FactorVIII in various proteolytically processed or otherwise truncated formsknown to those skilled in the art, wherein the various forms of FactorVIII possess procoagulant activity. Examples of factor VIII polypeptidesare those active factor VIII fragments and factor VIII derivativesdisclosed in Andersson et al., U.S. Pat. No. 4,749,780; Andersson etal., U.S. Pat. No. 4,877,614; Toole et al., U.S. Pat. No. 4,757,006;Toole, U.S. Pat. No. 4,868,112; Almstedt et al., U.S. Pat. No.5,661,008, all of which are incorporated herein by reference. The FactorVIII described in Almstedt et al. is made up of amino acids 1 to743 and1649 through 2332 of human factor VIII. This polypeptide sequence iscommercially known as rFVIII-SQ or REFACTO [r]. See also, Lind et al.,Euro. J. Biochem., 232:19-27 (1995). Other forms of truncated FVIII canalso be constructed in which the B-domain is generally deleted. In theAlmstedt et al. Factor VIII, the amino acids of the heavy chain,containing amino acids 1 through 740 of human Factor VIII and having amolecular weight of approximately 90 kD are connected to the amino acidsof the light chain, containing amino acids 1649 to 2332 of human FactorVIII and having a molecular weight of approximately 80 kD. The heavy andlight chains are connected by a linker peptide of from 2 to 15 aminoacids, for example a linker comprising lysine or arginine residues, oralternatively, linked by a metal ion bond. These other linkers anddifferent sized linkers could be used. See, also, Pipe and Kaufmann(109) for another Factor VIII variant which was genetically engineeredby deletion of residues 794-1689 so that the A2 domain is covalentlylinked to the light chain. Missense mutations at thrombin and activatedprotein C inactivation cleavage sites provide resistance to proteolysis,resulting in a single-chain protein that has maximal activity after asingle cleavage after arginine-372.

A human factor VIII cDNA nucleotide and predicted amino acid sequencesare shown in U.S. Pat. No. 6,180,371. Factor VIII is synthesized as anapproximately 300 kDa single chain protein with internal sequencehomology that defines the “domain” sequence NH₂-A1-A2-B-A3-C1-C2-COOH.In U.S. Pat. No. 6,180,371, factor VIII domains include the followingamino acid residues, when the sequences are aligned with the human aminoacid sequence set forth in that patent: A1, residues Ala1-Arg372; A2,residues Ser373-Arg740; B, residues Ser741-Arg1648; A3, residuesSer1690-Ile2032; C1, residues Arg2033-Asn2172; C2, residuesSer2173-Tyr2332. The A3-C1-C2 sequence includes residuesSer1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, isusually referred to as the factor VIII light chain activation peptide.Factor VIII is proteolytically activated by thrombin or factor Xa, whichdissociates it from von Willebrand factor, forming factor VIIIa, whichhas procoagulant function. The biological function of factor VIIIa is toincrease the catalytic efficiency of factor IXa toward factor Xactivation by several orders of magnitude. Thrombin-activated factorVIIIa is a 160 kDa A1/A2/A3 -C1 -C2 heterotrimer that forms a complexwith factor IXa and factor X on the surface of platelets or monocytes oron other surfaces.

The heavy chain of factor VIII contains the A1 and A2 domains and mayalso contain part or all of the B domain. (The heavy chain of B-domaindeleted factor VIII contains two domains, A1 and A2, and may contain asmall part of the B-domain.) The light chain of factor VIII containsthree domains, A3, C1, and C2.

Factor VIII Pharmaceutical Compositions

Pharmaceutical compositions containing disulfide-stabilized factor VIII,alone or in combination with appropriate pharmaceutical stabilizationcompounds, delivery vehicles, and/or carrier vehicles, may be preparedaccording to known methods, such as those described in Remington'sPharmaceutical Sciences by E. W. Martin, incorporated herein byreference. Pharmaceutical compositions may contain factor VIIIpolypeptide, nucleic acid coding for factor VIII, or the like.

In one preferred embodiment, the preferred carriers or delivery vehiclesfor intravenous infusion are physiological saline or phosphate bufferedsaline that may include sugars.

In another preferred embodiment, suitable stabilization compounds,delivery vehicles, and carrier vehicles include but are not limited toother human or animal proteins such as albumin.

Phospholipid vesicles or liposomal suspensions are also preferred aspharmaceutically acceptable carriers or delivery vehicles. These can beprepared according to methods known to those skilled in the art and cancontain, for example, phosphatidylserine/-phosphatidylcholine or othercompositions of phospholipids or detergents that together impart anegative charge to the surface, since factor VIII binds to negativelycharged phospholipid membranes. Liposomes may be prepared by dissolvingappropriate lipid(s) (such as stearoyl phosphatidylethanolamine,stearoyl phosphatidylcholine, arachadoylphosphatidyl choline, andcholesterol) in an inorganic solvent that is then evaporated, leavingbehind a thin film of dried lipid on the surface of the container. Anaqueous solution of the factor VIII is then introduced into thecontainer. The solution is mixed to free lipid material from the sidesof the container and to disperse lipid aggregates, thereby forming theliposomal suspension.

The factor VIII can be combined with other suitable stabilizationcompounds, delivery vehicles, and/or carrier vehicles, including vitaminK-dependent clotting factors, tissue factor, von Willebrand factor (vWf)or a fragment of vWF that contains the factor VIII binding site, andpolysaccharides such as sucrose.

Factor VIII can be stored bound to vWf to increase the half-life andshelf-life of the molecule. Additionally, lyophilization of factor VIIIcan improve the yields of active molecules in the presence of vWf.Methods for storage of factor VIII include: lyophilization of factorVIII in a partially-purified state (as a factor VIII “concentrate” thatis infused without further purification), andimmunoaffinity-purification of factor VIII and lyophilization in thepresence of albumin, which stabilizes the factor VIII. Factor VIII canalso be prepared by a process that uses sucrose as a stabilizer in thefinal container in the place of albumin. It is preferred that FactorVIII be prepared by a process that doesn't include any plasma or plasmaproteins. (See, e.g., Boedeker (111) and Cho et al., U.S. Pat. No.6,358,703 B1).

Additionally, factor VIII has been indefinitely stable at 40° C. in 0.6MNaCl, 20 mM MES, and 5 mM CaCl₂ at pH 6.0 and also can be stored frozenin these buffers and thawed with minimal loss of activity.

Methods of Treatment

Factor VIII is used to prevent, treat or ameliorate uncontrolledbleeding due to factor VIII deficiency (e.g., intraarticular,intracranial, or gastrointestinal hemorrhage) in subjects such ashemophiliacs with and without inhibitory antibodies and patients withacquired factor VIII deficiency due to the development of inhibitoryantibodies (51). The preferred subjects are mammals, most preferablyhumans. The active materials are preferably administered intravenously.

“Factor VIII deficiency,” as used herein, includes deficiency inclotting activity caused by production of defective factor VIII, byinadequate or no production of factor VIII, or by partial or totalinhibition of factor VIII by inhibitors. Hemophilia A is a type offactor VIII deficiency resulting from a defect in an X-linked gene andthe absence or deficiency of the factor VIII protein it encodes.

Additionally, factor VIII can be administered by transplant of cellsgenetically engineered to produce the factor VIII or by implantation ofa device containing such cells, as described above.

In a preferred embodiment, pharmaceutical compositions of factor VIIIalone or in combination with stabilizers, delivery vehicles, and/orcarriers are infused into patients intravenously.

The treatment dosages of factor VIII composition that must beadministered to a patient in need of such treatment will vary dependingon the severity of the factor VIII deficiency. Generally, dosage levelis adjusted in frequency, duration, and units in keeping with theseverity and duration of each patient's bleeding episode. Accordingly,the factor VIII is included in the pharmaceutically acceptable carrier,delivery vehicle, or stabilizer in an amount sufficient to deliver to apatient a therapeutically effective amount of the factor VIII to stopbleeding, as measured by standard clotting assays.

Factor VIII is classically defmed as that substance present in normalblood plasma that corrects the clotting defect in plasma derived fromindividuals with hemophilia A. The coagulant activity in vitro ofpurified and partially-purified forms of factor VIII is used tocalculate the dose of factor VIII for infusions in human patients and isa reliable indicator of activity recovered from patient plasma and ofcorrection of the in vivo bleeding defect. See, e.g., Lusher, J. M., etal., New. Engl. J. Med. 328:453-459 (1993); Pittman, D. D., et al.,Blood 79:389-397 (1992), and Brinkhous et al., Proc. Natl. Acad. Sci.USA, 82:8752-8755 (1985).

Usually, the desired plasma factor VIII level to be achieved in thepatient through administration of the hybrid or hybrid equivalent factorVIII is in the range of 30-100% of the normal plasma level. Typicaldosages for treatment of hemorrhage from hemophilia A with Factor VIIIare 25-50 units/kg of body weight. One unit=the normal amount of VIII in1 ml of citrated normal human plasma. See, e.g., Roberts, H R andHoffman, M. Hemophilia A and Hemophilia B. in Williams Hematology, 6thedition. eds E Beutler, M A Lichtman, B S Coller, T J Kipps and USeligson. McGraw-Hill, NY. 2001. In a preferred mode of administrationof factor VIII of the invention, which is expected to have increasedstability due to the introduction of one or more cysteine residues, thecomposition is given intravenously at a preferred dosage in the rangefrom about 0.1 to 80 units/kg body weight, more preferably in a range of0.5 to 50 units/kg body weight, more preferably in a range of 1.0-50units/kg body weight, and most preferably at a dosage of 2.0-40 units/kgbody weight; the interval frequency is in the range from about 8 to 24hours (in severely affected hemophiliacs); and the duration of treatmentin days is in the range from 1 to 10 days or until the bleeding episodeis resolved. See, e.g., Roberts, H. R., and M. R. Jones, “Hemophilia andRelated Conditions-Congenital Deficiencies of Prothrombin (Factor II,Factor V, and Factors VII to XII),” Ch. 153, 1453-1474, 1459-1460, inHematology, Williams, W. J., et al., ed. (1990). Patients withinhibitors may require more factor VIII of the invention, or patientsmay require less factor VIII of the invention because of its greaterstability than human factor VIII. In treatment with factor VIII, theamount of factor VIII infused is defined by the one-stage factor VIIIcoagulation assay and, in selected instances, in vivo recovery isdetermined by measuring the factor VIII in the patient's plasma afterinfusion. It is to be understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration and other ranges set forth herein are exemplaryonly and are not intended to limit the scope or practice of the claimedinvention.

Treatment can take the form of a single intravenous administration ofthe composition or periodic or continuous administration over anextended period of time, as required. Alternatively, factor VIII can beadministered subcutaneously or orally with liposomes in one or severaldoses at varying intervals of time.

Hybrid animal/human factor VIII of the invention can be used to treatuncontrolled bleeding due to factor VIII deficiency in hemophiliacs whohave developed antibodies to human factor VIII. In this case, coagulantactivity that is superior to that of natural human or animal factor VIIIalone is not necessary. Coagulant activity that is inferior to that ofnatural human factor VIII (i.e., less than 3,000 units/mg) will beuseful if that activity is not neutralized by antibodies in thepatient's plasma.

Factor VIII can also be delivered by gene therapy. The generalprinciples for this type of therapy are known to those skilled in theart and have been reviewed in the literature (e.g. 52, 53, 57). Variousstrategies have been utilized to deliver factors VIII and IX by genetherapy and many ofthese may be appropriate for delivery of factor VIIIthat is modified by the addition of engineered disulfide bonds.Following is a summary of the various approaches that could be utilized.

By far the largest volume of experience has been with retroviralvectors. An example of the extant peer-reviewed and publishedpreclinical data using retroviral vectors to treat hemophilia comes fromKay et al (58), who prepared a retroviral vector expressing canine FIXand infused it into the portal vein of hemophilic dogs that hadundergone partial hepatectomy. They were able to demonstrate long-termexpression of canine FIX (>2 years) but at levels that were far too lowto be therapeutic in humans.

Another approach, also for hemophilia B, makes use of an AAV vector. AAVvectors in present use are engineered from a parvovirus, AAV serotype 2,with a small (4.7 kb) single stranded DNA genome. Many individuals areinfected with the wild-type virus as children, but infection is notassociated with any known illness. The virus is naturallyreplication-defective, and the engineered vector is completely devoid ofviral coding sequences. Preclinical studies by several groups have shownthat AAV vectors can direct sustained expression of a transgeneintroduced into skeletal muscle, liver, or central nervous system(62-64). In the case of FIX, experiments in mice have resulted inexpression levels of 250 to 350 ng/mL (5% to 7% of nonnal circulatinglevels), whereas similar experiments in hemophilic dogs resulted inlevels of 70 to 80 ng/mL (approx. 1.5% of normal levels (65, 66)).

Efforts are also underway to extend the use of a liver-directed AAVapproach to FVIII, but the size of the transgene presents a problem inthis case, because AAV vectors cannot accommodate inserts above 5 kb andthe B domain-deleted FVIII cDNA (without promoter, intron, orviral-inverted terminal repeats) is 4.4 kb. Because of these sizeconstraints, several novel strategies have been devised to allowexpression of FVIII from an AAV vector (76, 77, 78).

A different approach that is currently being evaluated for treatment ofhemophilia A is ex vivo introduction of a plasmid expressingB-domain-deleted (BDD) FVIII into autologous fibroblasts, which are thenreimplanted on the omentum. In this strategy, a skin biopsy fiom thepatient serves as a source of autologous fibroblasts, which are thentransfected by electroporation with a plasmid expressing BDD FVIII and aselectable marker. After transfection, FVIII-expressing cells areselected, expanded, and reimplanted on the omentum in a laparoscopicprocedure (using on the order of 10⁸ to 10⁹ cells) (107).

Adenoviral vectors have several attractive features as gene deliveryvehicles, including ease of preparation and efficient transduction ofthe liver after introduction of vector into the peripheral circulation.These characteristics were exploited by Kay et al (80) to obtainhigh-level expression of canine FIX in hemophilic dogs as an early proofof principle for this approach. Several important insights aboutadenoviral vectors have been gained through the work of Connelly andcolleagues (83-87), who have explored the use of earlier generationadenoviral vectors as an approach to treating hemophilia A. Using anadenoviral vector expressing B domain-deleted FVIII, these workers wereable to demonstrate phenotypic correction of the-bleeding diathesis inmice with hemophilia A (87). Levels of expression were initially >2000mU/mL and, as expected, declined gradually over 9 months to approx. 100mU/mL.

Lentiviral vectors (101), a newer gene delivery vehicle based on HIV,have also been shown to transduce liver, muscle, and hematopoietic cellsand thus could potentially be used for gene therapy for hemophilia. Workpublished by Kafri et al (102) demonstrated stable expression (22 weeks)of a humanized GFP after direct intraparenchymal injection into liver ofa lentiviral vector.

Okoli et al (106) have presented a preliminary report in which FIXplasmid DNA contained within a chitosan DNA nanosphere is embeddedwithin gelatin cubes and fed to mice at a dose of 25 g plasmid in asingle treatment. Treated mice showed levels of 45 ng/mL (approx. 1%normal plasma levels), although levels gradually declined toundetectable over a 14-day period.

Phase I clinical trials in humans are underway or in late planningstages for retroviral vectors, AAV vectors, transfected plasmids andadenoviral vectors.

As will be obvious to those of skill in the art, similar methods may beused for the administration of entities other than factor VIII such asfactor V, prothrombin, factor XII, HGFA (hepatocyte growth factoractivator), and PHBP (plasma hyaluronin binding protein).

The following examples illustrate certain embodiments of the presentinvention, but should not be construed as limiting its scope in any way.Certain modifications and variations will be apparent to those skilledin the art from the teachings of the foregoing disclosure and thefollowing examples, and these are intended to be encompassed by thespirit and scope of the invention.

EXAMPLE 1 Factor V

In one embodiment of the present invention, one may engineer intorecombinant FV mutants a disulfide bond between the A2 and the A1 or A3domains such that dissociation of the A2 domain is prevented. Neitherthe x-ray crystal structure nor NMR structure of FVa is known. However,as noted above, the present invention is not limited to use with suchstructures and may be applied to homology models.

Accordingly, the computer program MODIP (19), which employs thealgorithm of Sowdhamini, was applied to the Pellequer homology model ofFVa (20). As noted above, MODIP predicts sites for the introduction ofdisulfide bridges and provides grades (A, B, C) for each prediction.Grade A sites are those predicted to be most optimal for theestablishment of disulfide bridges, while grade B and grade C sites areprogressively less ideal.

For the Pellequer FVa model, no grade A sites were predicted at eitherthe A1-A2 or A2-A3 interfaces, a single grade B site was predicted, andseveral grade C sites were predicted. Of the grade C sites predicted,MODIP indicated five sites to be the most ideal: His609 - Glu1691(A2-A3) Leu238 - Gln590 (A1-A2) His253 - Asp469 (A1-A2) Ala257 - Met618(A1-A2) Leu283 - Met618 (A1-A2)

It was noted that of these, the pair 609-1691 aligned with residuesTyr664-Thr1826 in Factor VIII.

Visual inspection of the predicted grade B and C sites usingcomputational graphics analysis showed the grade B site to be unusable.Next, a version of the FVa homology model further including a disulfidebridge was constructed for each of the five best grade C sites. This wasdone using the Xfit computer program, with refinement being provided bythe X-PLOR computer program using the Charm22 all atoms force field.

After refinement, the modeled disulfide bonds were analyzed for optimaldisulfide geometry. Cys609-Cys1691 provided the best potential geometryfor a disulfide bond in FV with r_(ss)=2.02 Å, χ_(xx)=80.9°, and thelowest Van Der Waals gas phase energy of the five sites. The second bestsite was Leu238-Gln590, with r_(ss)=2.03 Å and χ_(ss)=−111.6°. Thus,these two sites were chosen for initial attempts to create disulfidebonds using site-directed mutagenesis.

Next a plasmid pED-FV containing full-length FV cDNA was obtained. Thefull-length FV cDNA in the plasmid pED-FV was then removed by digestionwith SalI and inserted into a modified pUC119 plasmid. A fragment of theFV cDNA was next created with PCR using a 5′ primer that created a BamHIsite at nt4641 (FV cDNA numbering; nt=nucleotide) and a 3′ primer thatretained the BamHI site at nt6014 while removing the BamHI site atnt5975. The primers used are shown below, where underlining indicatesmutation and boldface indicates a codon or restriction site of interest:5′-primer (4641 site) 5′-CAC G GATCC TACAGATTACATTGAGATCA-3′ 3′-primer(5975 removal, retain 6014)5′-GTCTGGATCCCTGTGATTATGACTTCCTTTTGCATGTCCACCTGA AT CCAAG-3′

The pUC119-FV was digested with Bam-HI (cutting at nt2601, 5975 and 6014in FV cDNA numbering). The new PCR fragment was inserted between theBamHI sites in pUC119-FV between nt2601 and 6014. These steps resultedin the removal of nt2602 to 4641 (coding sequence for residues 812 to1491) creating a construct encoding a B-domainless FV designated FVΔB.

This FVΔB gene construct was inserted into the expression vectorpcDNA3.1+ from Invitrogen (Carlsbad, Calif.). Then, using the StratageneQuikchange PCR mutagenesis kit (La Jolla, Calif.) and FVΔB, Ser2183 wasmutated to Ala (changing codon AGT to GCC) to prevent glycosylation atAsin2181, yielding the mutant 2183A-FVΔB. This mutation was made toavoid FV heterogeneity due to incomplete glycosylation at Asn2181 whichgives two species of FV that differ in certain functional properties(25, 26). All subsequent mutations were made using this B-domainless,Ser2183A mutant. In some embodiments, this step maybe eliminated.

At the same time, the Stratagene Quikchange PCR mutagenesis kit was usedto place coding for cysteine residues by the addition of four mutagenicprimers. The following pairs were made: Leu238Cys:Gln590Cys(Cys238/Cys590), and His609Cys:Glu1691Cys (A2-SS-A3). Variants were alsomade with additional mutations of Arg506 and Arg679 to Gln (Gln506 orGln679) (Q506/Cys238/Cys590, Q506-A2-SS-A3 and Q506/Q679-A2-SS-A3). Themutagenesis primers used are shown below, where underlining indicatesmutation and boldface indicates a codon or restriction site of interest:Ser2183 - Ala Forward primer 5′-CATGGAATCAA GC TATTACACTTCGCC-3′ Reverseprimer 5′-GGCGAAGTGTAATA GC TTGATTCCATG-3′ Leu238 - Cys Forward5′-GGCCAGAAT GC TTCTCCATTC-3′ Reverse 5′-GAATGGAGAA GC ATTCTGGCC-3′Gln590 - Cys Forward 5′-GTGGGGACC TGT AATGAAATT-3′ Reverse 5′-AATTTCATTACA GGTCCCCAC-3′ His609 - Cys Forward 5′-CTATGGAAAGAGG TG TGAGGACACC-3′Reverse 5′-GGTGTCCTCA CA CCTCTTTCCATAG-3′ Glu1691-Cys Forward5′-GATCAGGGCCA TGT AGTCCTGGC-3′ Reverse 5′-GCCAGGACT ACA TGGCCCTGATC-3′Arg306 - Gln Forward 5′-CCAAAGAAAACC CA GAATCTTAAG-3′ Reverse5′-CTTAAGATTC TG GGTTTTCTTTGG-3′ Arg506 - Gln Forward 5′-CTGGACAGGCAAGGAATACAG-3′ Reverse 5′-CTGTATTCCTT GCCTGTCCAG-3′ Arg679 - Gln Forward5′-CATGGCTACACA GAAAATGCATG-3′ Reverse 5′-CATGCATTTTC T GTGTAGCCATG-3′

Plasmids containing each mutant were purified with the Qiafilter plasmidmidiprep kit from Qiagen, linearized and transfected into COS-1 cellsusing Superfect tranisfection reagent according to the manufacturer'sinstructions. More specifically, 1 μg of DNA was incubated in 60 μLvolume of DMEM/F12 media with 5 μL of Superfect reagent for ten minutes.Then 350 μL of DMEM/10% FBS/1 mM Glutamine was added and this mixturewas transferred to COS-1 cells (about 50% confluent) in wells of a24-well plate and incubated for 3 hours before washing and replenishingwith fresh media. Stable clones were selected using 0.8 mg/mL Geneticin(Gibco BRL, Rockville, Md.). Serum-free conditioned media containing0.05% BSA and 5 mM CaCl₂ was collected from COS-1 cells expressing eachFV mutant and was precipitated with 16% PEG 6000. Then the pellet wasredissolved in HBS (50 mM HEPES, 150 mM NaCl, pH 7.4) containing 5 mMCaCl₂, 2 mM benzamidine, 5 nM PPACK and 1 mM PMSF, dialyzed versus thesame buffer and purified using an anti-FV antibody column (24).Fractions containing FV were collected, concentrated and stored in HBSwith 0.1% BSA at −80 C.

FVa was quantified by activity and by ELISA assay after activation bythrombin. ELISA assays used Nunc Maxisorb plates coated with 10 μg/mlsheep-anti-FV from Affinity Biological (Hamilton, Ontario, Calif.) andblocked with Superblock from Pierce (Rockford, Ill.) with antigendetection by mouse anti-FV-light-chain monoclonal antibody (V59). FV (40nM) was activated with thrombin (0.5 nM) in HBS with 0.1% BSA and 5 mMCaCl₂ at 37° C. for 10 min and activation was stopped by the addition of1.1 molar equivalent of hirudin. FVa inactivation assays were performedusing FVa at 4 nM and APC at 2.5 nM with determination of residual FVausing prothrombinase assays as described (27). Inactivation of FVa wasmeasured as follows. A mixture of 1 nM FVa with 25 μM phospholipidvesicles was made in 50 mM HEPES, pH 7.4, 100 mM NaCl, 0.5% BSA, 5 μMCaCl₂, 0.1 mM MnCl₂ (called Ptase buffer). Inactivation was initiated bythe addition of APC. One μL aliquots were removed at time points andadded to 40 μL of 1.25 nM factor Xa with 25 μM phospholipid vesicles,followed by 10 μL of 3 μM prothrombin (final concentrations: 1 nM FXa,20 pM FVa, 25 μM phospholipid vesicles and 0.6 μM prothrombin). After2.5 min a 15 μL aliquot of this mixture was quenched by addition to 55μL TBS containing 10 mM EDTA, 0.5% BSA, pH 8.2. Chromogenic substrateCBS 34-47 was added and the amount of thrombin formation was assessed bymeasuring the change in absorbance at 405 nm.

For some studies, FXa or prothrombin was varied. For Xa titrations, amix of 3.34 pM FVa/FVai and 41.7 μM phospholipid vesicles in Ptasebuffer was aliquoted in 30 μL aliquots into wells of 96-well plate(polypropylene, V-well). 10 μL of Xa was added to each well in the samebuffer at various concentrations. At time=0, 10 μL of 1.5 μM prothrombin(FII) was added to all wells (final concentrations=2 pM FVa, 25 μM PLvesicles, 5-600 pM Xa, 0.3 μM FII). At time=12 min, the Ptase reactionwas stopped by removing 15 μL to a 96-well plate containing 55 μL TBScontaining 0.5% BSA, 10 mM EDTA at pH 8.2. Next, the amount of thrombinformed was measured with the chromogenic substrate CBS 34-47. Forprothrombin, 20 μL of mix containing 125 pM Xa, 1.25 nM FVa/FVai, and31.25 μM PL vesicles in Ptase buffer was aliquoted into wells of 96-wellV-well plate. At time=0, 5 μL FII at varying concentrations (finalconcentration 100 pM Xa, 1 nM FVa, 25 μM PL, 25-1500 nM FII) was added.At time=2:30, the reaction was stopped by removing 15 μL to 55 μL EDTAbuffer as above. Thrombin was measured as above.

SDS-PAGE was then performed with Novex 4-12% Bis-Tris gradient gels withMOPS buffer (Invitrogen, Carlsbad, Calif.). 50 ng protein was loaded perlane. The proteins were then transferred to Millipore PVDF membranes,and immunoblots were developed with monoclonal anti-FV-light chainantibodies, AHV-5112 or V59, and rabbit polyclonal anti-FV-heavy chainantibodies (24). More specifically, membranes were blocked with TBS, 1%Casein, and 2 mM CaCl₂. Antibodies were diluted in the same buffer. Theprimary antibody was the respective anti-FV antibody, and the secondaryantibody was biotinylated goat anti-mouse IgG or biotinylated donkeyanti-rabbit IgG from Pierce. Visualization was then performed withstreptavidin-conjugated alkaline phosphatase and 1-step NBT/BCIPsubstrate (also from Pierce). For the FV species that were produced andpurified, yields of pure FV ranged from 5 to 25 μg/L of conditionedmedia. Based on silver-stained SDS-PAGE, we estimated the purity of themutants to range from 70% to 90%.

As is known in the art, the FVa light chain normally gives a doublet onSDS-PAGE due to heterogeneity created by incomplete glycosylation atAsn2181. Mutation of Ser2183 to Ala eliminates this glycosylation site(28). Immunoblots confirmed that all our recombinant FV molecules had anapparent molecular weight of 188 kDa, consistent with deletion ofresidues 812 to 1491. Immunoblots further confirmed that the wild typerecombinant FVa formed a light chain doublet, whereas all other Fvamutants carrying the Z183A mutation had only a single light chain band.

To demonstrate the desired interdomain disulfide bonds in the mutant FVproteins containing two engineered cysteine residues, immunoblots of FVaand APC-treated FVai (where “i” indicates inactivated) were run. FIG. 1shows schemes representing the primary sequences of FVΔB, FVa (formedupon thrombin activation), and FVai (inactivated by APC cleavages).

Immunoblots using a polyclonal anti-FV heavy chain antibody demonstratedthat introduction of Cys238/Cys590 mutations into FV or Q506-FV did notdetectably link the A1 and A2 domains although these species had normalFVa activity, leading us to conclude that no disulfide bond was formedbetween these cysteines.

If FV mutants containing Cys609 and Cys1691 generate a new disulfidebond between the A2 and A3 domains as depicted in FIG. 1C, it would linkthe FVa heavy and light chains. In this case, in immunoblots of FVa, thedisulfide-bonded species would appear at a molecular weightcorresponding to the additive molecular weights of the heavy and lightchains, and following APC cleavages at Arg506, Arg306 and Arg679 thatnormally cause complete FVa inactivation, the light chain of FVai wouldremain cross-linked to the C-terminal fragment of the A2 domain (A2c,residues 507 to 679).

Indeed such results were obtained. In immunoblots developed with anti-FVlight chain antibodies (FIG. 2A), lanes 1 and 2 containing 2183A-FVa and2183A-FVai both showed a normal light chain at the expected molecularweight (69 kDa), whereas in lane 3, the mutant containing Cys609/Cys1691-FVa showed a predominant band predicted for cross-linked lightchain and heavy chain (158 kDa). Thus, FV mutants containing these twoCys residues are justifiably designated “A2-SS-A3”.

Lane 4 demonstrated that APC-treated A2-SS-A3-FVai gave a predominantband corresponding to the mobility predicted for the light chaincross-linked to the A2c fragment (92 kDa). A fainter band slightly abovethis band correlated with a band predicted for heavy chain cleaved atArg506 but not Arg679, resulting in the fragment 507 to 709 (101 kDa).Lanes 5 and 6 (FIG. 2A) contained Q506-A2-SS-A3-FVa andQ506-A2-SS-A3-FVai. In these species, Arg506 cleavage cannot take placesuch that in Q506-A2-SS-A3-FVai (lane 6) the light chain remainedcross-linked to the entire A2 domain (with or without its smallC-terminal tail of residues 680-709). Indeed, the observed highermolecular weight band (lane 6) corresponded to the light chaincross-linked to the A2 domain (130 kDa). Lanes 7 through 12 of FIG. 2Acontained samples parallel to those of lanes 1 through 6, which werereduced using DTT. Lanes 7-12 show that, following reduction, thevarious higher molecular weight cross-linked species disappeared andnormal light chain bands appeared, proving that the higher molecularweight light chain-containing species seen in lanes 3-6 (FIG. 2A) wereindeed the result of disulfide cross-lhiks between light and heavychains.

Additional proof for covalent cross-links between FVa heavy and lightchains in A2-SS-A3 mutants containing Cys609/Cys1691 came fromimmunoblot analyses using anti-FV heavy chain antibodies that showed,under non-reducing conditions, the same new bands visualized inimmunoblots developed using anti-FV light chain antibodies. For example,in FIG. 2B such immunoblots of A2-SS-A3-FVa and A2-SS-A3-FVai as well asQ506-A2-SS-A3-FVa and Q506-A2-SS-A3-FVai under non-reducing conditionsgave bands predicted to represent the same cross-linked speciesvisualized in immunoblots developed using anti-FV light chain antibodiesFIG. 2B. Lanes 1 and 5 (FIG. 2B) both showed bands corresponding tolight chain cross-linked to heavy chain that co-migrated with that seenin FIG. 2B, lane 3 (157 kDa). Lane 2 in FIG. 2B showed a bandcorresponding to the light chain cross-linked to the A2c fragment,co-migrating with a band seen in lane 4 of FIG. 2A (102 kDa). Lane 6 inFIG. 2B showed a band corresponding to the light chain cross-linked tothe A2 domain, equivalent to a band seen in lane 6 of FIG. 2A (132 kDa).

Finally, free A2-C terminus fragment (24 kDa) and A2 (63 kDa) fragmentwere not visible in the non-reduced lanes 2 and 6, respectively, butwere visible in the reduced lanes 4 and 8, indicating that thesefragments were released from the disulfide-linked species uponreduction.

Immunoblot analyses of Q506-A2-SS-A3 FVa and Q506/Q679-A2-SS-A3-FVashowed that there was a small amount of free light chain that was notcross-linked to heavy chain (FIG. 2), indicating that disulfidecross-linking in the A2-SS-A3-FVa mutants was not 100% complete.Densitometry analysis of these non-reduced immunoblots showed that, onaverage, about ten percent of the Q506-A2-SS-A3-FVa molecules lackeddisulfide cross-links.

As alluded to above, FIG. 1A is a schematic of the primary sequence ofFVΔB with the locations of the different domains indicated. Theschematic of FIG. 1 b shows activated FVΔB (FVa), a heterodimer of theN-terminal heavy chain and the C-terminal light chain associated in thepresence of Ca²⁺ ions. Arrows indicate sites of cleavage in FVa by APC.The schematic of FIG. 1C shows the cleavage fragments produced uponinactivation of FVa (FVai) by APC, and further shows the sites of thecysteine mutations that did (His609-Glu1691) and did not (Leu238-Gln590)result in disulfide bond formation.

EXAMPLE 2 Factor VIII

As is known in the art, there are a number of similarities betweenFactor V and Factor VIII. More specifically, Factors V and VIII havesimilar gene structures, have highly homologous amino acid sequences anddomain structures, are both activated by highly specific cleavages bythrombin, and both are inactivated by limited proteolysis by activatedprotein C (APC). Accordingly, one may engineer into recombinant FVIIIdisulfide bonds between the A2 and the A1 or A3 domains using a methodsimilar to that disclosed above concerning FV. As is known in the art,FVIIIa is thermodynamically unstable because the A2 domain canspontaneously disassociate. As shown in FIG. 3, placement of a disulfidebond between the A2 and the A1 or A3 domains of FVIIIa has the advantageof preventing this dissociation.

Like FVa, neither the x-ray crystal nor NMR structure of FVIIIa isknown. However, as noted above, the present invention is not limited touse such structures and may be applied to homology models.

As a first step in engineering a disulfide bond between the A2 and theA1 or A3 domains of FVIIIa, the computer program MODIP, which employsthe algorithm of Sowdhamini, was applied to the Pemberton et al. (54)homology model of the A domains of FVIIIa. As noted above, MODIPpredicts sites for the introduction of disulfide bridges and providesgrades (A, B, C) for each prediction. Grade A sites are those predictedto be most optimal for the establishment of disulfide bridges, whilegrade B and grade C sites are progressively less ideal. For thePemberton FVIIIa model fifteen sites were predicted: Grade A: Met 662 -Asp 1828 (A2-A3) Grade B: Ser 268 - Phe 673 (A1-A2) Ile 312 - Pro 672(A1-A2) Ser 313 - Ala 644 (A1-A2) Met 662 - Lys 1827 (A2-A3) Tyr 664 -Thr 1826 (A2-A3) Grade C: Pro 264 - Gln 645 (A1-A2) Arg 282 - Thr 522(A1-A2) Ser 285 - Phe 673 (A1-A2) His 311 - Phe 673 (A1-A2) Ser 314 -Ala 644 (A1-A2) Ser 314 - Gln 645 (A1-A2) Val 663 - Glu 1829 (A2-A3) Asn694 - Pro 1980 (A2-A3) Ser 695 - Glu 1844 (A2-A3)

Of these, the pair Tyr 664-Thr 1826 was noticed to be in a positionhomologous to the pair His609-Glu1691 in FVa. As noted above, adisulfide bridge may be successfully engineered into FV by placingcoding for cysteine residues at positions 609 and 1691.

Similar to the method described above for FV, visual inspection of thesepairs was preformed using computational graphics analysis. As a resultof this analysis, three of the proposed pairs were chosen for furtherinvestigation: Met 662-Asp 1828, Tyr 664-Thr 1826 and Ser 313-Ala 644.For each of these three sites, a version of the FVIIIa model furtherincluding a disulfide bridge at the appropriate location was constructedusing the Xfit computer program, with refinement being provided by theX-PLOR computer program using the Charm22 all atoms force field. Afterrefinement, the modeled disulfide bonds were ranked in the order givenabove with Cys 662-Cys 1828 providing the best potential geometry for adisulfide bond. It was chosen to make this mutant and the mutant Cys664-Cys1826 in recombinant factor VIII in a maiiner analogous to thatdescribed above with reference to FV.

A FVIII expression plasmid (p25D) was obtained from Bayer Corporation.This plasmid expresses B-domain deleted FVIII in which residues 744 to1637 from the B domain are deleted.

Next, using the Stratagene Quikchange PCR mutagenesis and the mutantFVIII, two cysteine residues were inserted to permit the creation of adisulfide bond by the addition of four mutagenic primers at the sametime. The following two pairs were made: Met662Cys:Asp1828Cys andTyr664Cys:Thr1826Cys. The mutagenesis primers used are shown below,where underlining indicates mutation and boldface indicates a codon orrestriction site of interest: Met662 - Cys Forward 5′-CCTTCAAACACAAA TGCGTCTATGAAGACACACTCACC-3′ Reverse 5′-GGTGAGTGTGTCTTCATAGAC GCATTTGTGTTTGAAGG-3′ Asp1828 - Cys Forward 5′-GGCACCCACTAAA TGTGAGTTTGACTGCAAAGC-3′ Reverse 5′-GCTTTGCAGTCAAACTCA CA TTTAGTGGGTGCC-3′Tyr664 - Cys Forward 5′-CACAAAATGGTCTG TGAAGACACACTCACCC-3′ Reverse5′-GGGTGAGTGTGTCTTCAC AGAGGATTTTGTG-3′ Thr1826 - Cys Forward5′-CATATGGCACCC TG TAAAGATGAGTTTGACTGC-3′ Reverse 3′-GCAGTCAAACTCATCTTTACA GGGTGCCATATG-3′

The Tyr664-Cys reverse primer shown above was the actual sequence usedbut the actual FVIII gene sequence at nucleotides 22 and 23 should be CCrather than GG. But the forward primer has the correct sequence and thecorrect sequence was selected for the final C664 mutant by DNAsequencing of the selected clones.

FIG. 4 is a schematic showing the expected action of APC upon mutantFVIII containing a disulfide bridge between sites Met 662-Asp 1828 orTyr 664-Thr 1826.

In some embodiments, variants may be made which additionally containmutations and/or deletions of APC cleavage sites Arg 336 and/or Arg 562in FVIII. Such additional mutations, as described in Kaufman and Pipe(109) and in U.S. Pat. Nos. 5,422,260, 5,250,421, 5,198,349(incorporated herein by reference), add additional stability to FVIII bymaking it more resistant to inactivation.

The nucleic acids encoding Factor VIII mutants may also be modified tocontain an increased number of preferred codons for human genes asdescribed, e.g., in Seed et al., U.S. Pat. No. 6,114,148.

Transient expression of wildtype and mutant p25D plasmid was tested inCOS-1 cells, K293 cells and BHK-21 cells using Superfect reagent andEffectene reagent, both from Qiagen. The Effectene reagent in K293 cellsgave the best results. Yields of recombinant FVIII ranged from 10 to 100mU/mL of conditioned media according to APTT activity assays and ELISA(Immubind FVIII ELISA, American Diagnostica). Conditioned media wascollected from transient transfections in 100 mm dishes in DMEM/F1 2media with 2% FBS and the media was concentrated 15-fold and dialyzedinto BEPES buffered saline/5 mM CaCl₂/0.1 mM MnCl₂, pH 7.4. Mocktransfection media was treated in the same manner and used as a negativecontrol.

Antigen concentration of recombinant FVIII was determined using theImmubind FVIII ELISA kit from American Diagnostica. The standard curveused was the purified FVIII concentrate provided with the kit (1unit=the FVIII contained in 1 mL of plasma). Activity was determinedwith an APTT assay with FVIII deficient plasma and the APTT reagentPlatelin LS as follows: 50 μL of FVIII deficient plasma (FVIIIdP, GeorgeKing Biomedical) was mixed with 50 μL Platelin LS (Organon Teknika) andincubated at 37° C. for three minutes. 5 μL of a FVIII sample was thenadded, immediately followed by 50 μL of HEPES buffered saline (0.15 MNaCl) with 0.5% BSA and 25 mM CaCl₂. Clotting time was measured in theDiagnostica Stago ST4 coagulometer. A FVIII standard curve was madeusing pooled normal human plasma (George King Biomedical), which isdefined to contain 1.0 unit/mL of FVIII. The APTT assay was sensitive tovery low levels of FVIII (<0.005 U/nL).

Using these measures of antigen and activity, relative specific activityof the three proteins was calculated (units (U) activity/units (U)antigen). The wildtype FVIII (B domain-deleted) had a relative specificactivity of 0.83. C662-C1828-FVIII had a relative specific activity of3.53 and C664-C1828-FVIII had a relative specific activity of 3.40.

The stability of thrombin-activated FVIIIa over time was followed usinga protocol described by Pipe et al (110) with some modification, inwhich FVIIIa at a concentration of about 500 mU/mL was generated by theaddition of thrombin which was then inactivated with a slight excess ofhirudin. Subsequently, aliquots of this mix were removed over time andimmediately assayed for FVJIIa activity in the APTT assay as describedabove. FIG. 5 shows the results of this assay with recombinant wildtypeFVIIIa and two recombinant mutants. The two double-cysteine mutants aremuch more stable over time than wildtype FVIIIa (as reflected in ashorter clotting time). The mock transfection control conditioned mediashowed essentially no coagulant activity in this assay and no change inactivity over the time course (data not shown).

The FVIII mutant produced may be stably transfected into cells. Thecells can be grown (or cultured) to permit expression of the FVIIImutants. The FVIII mutant produced may be isolated and purified. In amamier described above with reference to FV, immunoblots may beperformed to confirm the lack of the majority of the B domain (ifappropriate) and the presence of the engineered disulfide bonds.

EXAMPLE 3 Porcine-Human Hybrid Factor VIII

There exists in the art hybrid factor VIII molecules whose amino acidsequence derives from both human and non-human-animal (“non-human”)factor VIII coding sequences. Examples of such molecules may be found,for example in U.S. Pat. No. 6,180,371, incorporated herein byreference. According to the present invention, non-human/human hybridfactor VIII containing a disulfide bond between the hybrid's A2 and A1or A3 domains may be created. Like the above example, such a disulfidebond prevents dissociation of the A2 domain.

The creation of such hybrid molecules is largely analogous to theprocedure described above for non-hybrid FVIII. Firstly, a homologymodel of hybrid FVIIIa, for example, one comprised of a non-human A2domain and a heterodimer of des-A2 human factor VIIIa, may be obtainedor created. Alternately, an x-ray crystal structure may be obtained orcreated if such a structure exists or is capable of being created. TheMODIP computer program may next be run on the model or structure so asto receive from the program suggestions of sites for the formation of adisulfide bridge between the A2 and A1 or A3 domains of the hybrid.Alternately, predictive methods may be used as described above.

Next, visual inspection of one or more of the suggested sites may bepreformed using computational graphics analysis. As a result of thisanalysis, a number of proposed sites may be chosen for furtherinvestigation. For each of these sites, a version of the hybrid FVIIIamodel further including a disulfide bridge at the appropriate locationmay be constructed using the Xfit computer program, with refinementbeing provided by the X-PLOR computer program using the Charm22 allatoms force field. After refinement, the modeled disulfide bonds may beranked based on quality of potential geometry for a disulfide bond. Anumber of the suggested sites may then be chosen for attempted creationof mutant hybrid FVIII in a manner analogous to that described inreference to FV and non-hybrid FVIII above.

EXAMPLE 4 Prothrombin

As noted above, in the presence of thrombomodulin andphosphatidylserine/phosphatidylcholine phospholipid vesicles (PCPS),meizothrombin, as well as meizothrombin (des F1), are better activatorsof protein C than thrombin.

According to the present invention, a mutant prothrombin may be createdwhich includes a disulfide bond to stabilize prothrornbin'smeizothrombin (des F1) form, and to prevent the conversion ofmeizothrombin (des F1) to thrombin. Such a stable meizothrombin (des-F1)has potential application, for example, as an anticoagulant. It wasdecided to achieve this stabilization by placement of a disulfide bondbetween the Kringle 2 and protease domains of prothrombin as shown inFIG. 5.

First, the computer program MODIP was applied to the X-ray crystalstructure of a human thrombin complex of alpha-thrombin and fragment 2(55), and the X-ray crystal structure of bovine meizothrombin (des F1)(108) resulting in the following predicted sites in human prothrombin:Grade B: Asp261-Arg443 (KR2-protease) His205-Lys572 (KR2-protease) GradeC: Asp261-Lys567 (KR2-protease)

Next, visual inspection of one or more of the suggested sites may beperformed using computational graphics analysis. As a result of thisanalysis, a number of proposed sites may be chosen for furtherinvestigation. For each of these three sites, a meizothrombin (des F1)homology model including a disulfide bridge at the appropriate locationmay be constructed using the Xfit computer program, with refinementbeing provided by the X-PLOR computer program using the Charm22 allatoms force field. After refinement, the modeled disulfide bonds may beranked based on quality of potential geometry for a disulfide bond. Anumber of the suggested sites, or sites that have not yet beenidentified, may then be chosen for attempted creation of mutantprothrombin in a manner analogous to that described in reference to FVand non-hybrid FVIII above.

EXAMPLE 5 Factor XII, HGFA, and PHBP

As noted above, at least two forms of activated factor XII (FXIIa)exist, αFXIIa and FXIIa fragment. As also noted above, the FXIIafragment has the bulk of its N-terminal heavy chain fragment no longerassociated such that it no longer binds to surfaces but it is stillcatalytically active. According to the present invention, a disulfidebond can be placed that crosslinks this N-terminal heavy chain fragmentto the remainder of the molecule, causing it to retain its surfacebinding characteristics. It is expected that such a mutant stabilizedFXII could find pharmaceutical application as a coagulant.

A second FXII-like polypeptide is HGFA. HGFA activates hepatocyte growthfactor (HGF) within injured tissues, where HGF plays roles in tissuerepair. As noted above, cleavage of HGFA by kallikrein at Arg372 resultsin release of the N-terminal heavy chain, which, as in FXII, is involvedin surface binding. According to the present invention, a disulfide bondcan be placed to prevent the release of the N-terminal heavy chain. Itis suspected that a mutant HGFA so stabilized could be usedpharmaceutically to aid in tissue repair.

A third FXII-like polypeptide is PHBP. As noted above, PHBP activatesFVII, uPA, and tPA and has a structure homologous to HGFA. According tothe present invention, a disulfide bond could be placed to prevent therelease of the N-terminal heavy chain in PHBP. It is suspected that amutant PHBP so stabilized could be used pharmaceutically to promoteclotting via activation of FVII, uPA, and/or tPA.

No x-ray crystal or NMR structure exists for Factor XII, HGFA, or PBBP.However, homology models for these molecules, such as ones based on thex-ray crystal structure of urokinase, may be created or obtained. Usingsuch homology models, mutants may be created in a manner analogous tothat described above with reference to, for example, FV and FVIII.

EXAMPLE 6 Other Clotting Factors

As is known in the art, several plasma factors other than factors V andVIII are synthesized as a single polypeptide chain, contain multipleindependently folded domains, and are subject to limited proteolysisthat may result in separation of domains due to dissociation. As notedabove, the methods described herein may be used in all cases where onewishes to place a disulfide bond between two domains of a polypeptide.Accordingly, it should be apparent to those in the art that the methodsdescribed herein may be applied to a multitude of polypeptides,including many of the human and non-human clotting factors.

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Every reference cited here and throughout the application is herebyincorporated by reference in its entirety.

RAMIFICATIONS AND SCOPE

Changes may be made in the nature, composition, operation andarrangement of the various elements, steps and procedures describedherein without departing from the spirit and scope of the invention asdefimed in the following claims. Modifications of the above describedmodes for carrying out the invention that are obvious to those of skillin the fields of genetic engineering, virology, hematology, medicine,and related fields are intended to be within the scope of the followingclaims.

1. A method of stabilizing a polypeptide which is the product of asingle gene in nature by introducing one or more cysteines comprisingthe steps of: (a) obtaining or creating a three-dimensional structure ofsaid polypeptide; (b) predicting one or more sites for the introductionof one or more cysteines based on said structure; and (c) creating oneor more mutants of said polypeptide by introducing one or more cysteinesat one or more of said predicted sites; wherein the introduction of saidone or more cysteines permits the formation of at least oneintramolecular, interdomain disulfide bridge which increases thestability of the mutant polypeptide as compared to that of thepolypeptide which does not contain said introduced one or morecysteines.
 2. A method of stabilizing a polypeptide which is the productof a single gene in nature by introducing one or more cysteinescomprising the steps of: (a) obtaining or creating a three-dimensionalstructure based on homology modeling of said polypeptide; (b) predictingone or more sites for the introduction of one or more cysteines based onthe three dimensional structure; and (c) creating one or more mutants ofsaid polypeptide by introducing one or more cysteines at one or more ofsaid predicted sites; wherein the introduction of said one or morecysteines permits the formation of at least one intramolecular,interdomain disulfide bridge which increases the stability of the mutantpolypeptide as compared to that of the polypeptide which does notcontain said introduced one or more cysteines.
 3. The method of claim 1wherein said polypeptide is a plasma protein.
 4. The method of claim 1wherein said polypeptide is selected from the group consisting of FactorVIII, Factor V, prothrombin, Factor XII, hepatocyte growth factoractivator, and plasma hyaluronan binding protein.
 5. The method of claim4 wherein said polypeptide is Factor V or Factor VIII. 6.-48. (canceled)49. The method of claim 2 wherein said polypeptide is a plasma protein.50. The method of claim 2 wherein said polypeptide is selected from thegroup consisting of Factor VIII, Factor V, prothrombin, Factor XII,hepatocyte growth factor activator, and plasma hyaluronan bindingprotein.
 51. The method of claim 50 wherein said polypeptide is Factor Vor Factor VIII.